Electrophoresis is a useful technique for separating molecules including, but not limited to, carbohydrates, proteins, lipids, nucleic acids and derivatives thereof. Electrophoresis operates by separating molecules based on a combination of their molecular charge, their molecular weight and their overall structure.
Separation of molecules by electrophoresis has certain advantages over chromatographic techniques. Electrophoresis media is generally transparent, thereby facilitating visualization of separated molecules. Electrophoresis media is also generally of low flourescence, thereby enabling the use of fluorescent markers. Superior resolution of closely related molecules is achieved as compared to techniques such as Thin Layer Chromatography and High Performance Liquid Chromatography.
In electrophoresis, the molecules are separated as they pass through a media, the media being attached to a current source, such that the anode and cathode are located at opposite ends of the media. By applying a voltage potential difference between the anode and cathode, molecules loaded into the media to be separated migrate toward the electrodes based on their net charge, e.g. molecules with a net negative charge migrate toward the positive electrode or anode. The rate at which the charged molecules migrate is dependent on the charge density of the molecule, referred to as the charge/mass ratio, and the porosity of the electrophoresis media employed. Molecules with a high charge/mass ratio migrate faster toward the oppositely charged electrode than molecules with a low charge/mass ratio. Molecules with the same charge/mass ratio migrate as a discrete band during electrophoresis and are effectively separated from other molecules with different charge/mass ratios.
The rate at which molecules migrate during electrophoresis is also dependent on the restrictive nature of the media. Convection currents generated by uneven heating through the solution often result in significant band distortion and unsatisfactory separations. Consequently many electrophoresis systems utilize a porous electrophoresis media which is designed to minimize molecular diffusion and convection currents during electrophoretic separations.
Many different types of electrophoresis media have been described and used to maximize band resolution and minimize band distortion. Compounds used to formulate electrophoresis media include but are not limited to paper (cellulose), polyacrylamide, agarose, starch, cellulose acetate and sepharose. Compounds used to formulate electrophoresis media are hereinafter referred to as "matrix elements." In general, matrix elements are polymeric in nature and, under the proper conditions, form a porous gel that restricts the movement of molecules through the media during electrophoresis. Electrophoresis media is usually prepared as a porous gel either by the chemical cross-linking of long polymers through the addition of cross-linking reagents, as is the case with polyacrylamide, or by adjusting the component concentration to form a gelatinous network of polymers, as is the case when preparing agarose and starch gels. In either case, the method by which an electrophoresis media is formed from its corresponding matrix element(s) is hereinafter referred to as "curing."
The essential feature of all electrophoresis media is that during electrophoresis, the stable media functions effectively as a molecular sieve so that molecules can be separated based on a combination of their hydrodynamic radii or molecular size and their charge/mass ratio.
In order for the molecular sieving to be effective in separating molecules based on their molecular size, the relationship between the effective pore size of the media and the size of the molecules being separated must be considered. If the media pore size is significantly larger than the size of the molecules being separated, the electrophoretic separation will be based largely on differences in the charge and the effect of molecular sieving will be minimal. Because of their chemical nature, different electrophoresis media possess different nominal pore sizes. As a result, different media have been found to be more appropriate for selected separations. For example, agarose gels have a relatively large pore size and are therefore most effective for separating large molecules such as large nucleic acids. Because of their large pore size, agarose gels are generally not used for protein separations. Effective protein separations require an electrophoresis media having a pore size that closely approximates the protein's smaller molecular size. Electrophoretic protein separations are generally performed using uniform polyacrylamide gels prepared using 10-20% polyacrylamide concentrations. These gels result in a bore size of 100,000-1,000,000 Daltons that effectively separate proteins in the 25,000-200,000 Dalton range.
In order to effectively separate proteins smaller than 25,000 Daltons or small oligonucleotides by electrophoresis, polyacrylamide gels having a concentration greater than 15% are required.
Electrophoresis techniques have also been developed using high percentage (15-40%) polyacrylamide gels for separating carbohydrates through the use of fluorophore labels. Fluorophore assisted carbohydrate electrophoresis permits the electrophoretic separation of a complex mixture of carbohydrates into distinct bands on a gel. Prior to electrophoresis, a carbohydrate mixture for analysis is treated with a fluorophore label that combines with the reducing end of the carbohydrates for analysis. The fluorophore label permits the quantitative measurement of the labeled carbohydrates by fluorescence. The fluorophore label is either itself charged or coupled to a species that imparts a charge on an otherwise uncharged fluorophore. Thus, the fluorophore label not only fluorescently tags the carbohydrates, it also imparts an ionic charge that permits otherwise uncharged carbohydrates to migrate in an electric field. The fluorophore assisted carbohydrate electrophoresis technique is described in detail in U.S. Pat. Nos. 4,317,480, 4,874,492 and in co-pending U.S. patent application Ser. No. 07/317,480, filed Feb. 14, 1989, all of which are incorporated herein by reference. The application of high percent acrylamide gels for separating small proteins, small oligonucleotides and carbohydrates have created a commercial demand for stable high percentage polyacrylamide gels.
Polyacrylamide gels require special preparation. Polyacrylamide gels are usually "poured" as a thin slab within a chamber or space formed between two plates of glass or plastic. This arrangement is called a gel cassette. The thickness of the gel is defined by the distance between the plates of the cassette which in turn is defined by the thickness of the two spacers that extend from the top to the bottom of the cassette along the outside edges. The thickness of the spacers controls the thickness of the electrophoresis media formed within the cassette and is generally between 0.25 mm to 3 mm in thickness. Depending on the application, the resolution of molecular separations generally increases with decreasing media thickness. In addition to the spacers, the cassette also contains a "comb" that is used to form chambers within the media. The number of chambers formed in the media by the comb can range from 1 to as many as 60 or greater, depending on the design of the comb. Prior to electrophoresis, the comb is removed. The samples are then applied into the wells formed by the comb.
In order to obtain optimal separation and resolution of molecules in the sample, polyacrylamide gels are often prepared with regions that contain different effective pore sizes. This is often referred to as a stacking buffer system, also known as "moving boundary electrophoresis." The stacking buffer system is only one example of multiple component electrophoresis media systems.
A stacking buffer system uses techniques known to work with protein and DNA fragments and is described in the book "Gel electrophoresis of proteins: a practical approach," edited by B. D. Hames and D. Rickwood, published by IRL Press.
In a stacking buffer system, a low percent acrylamide solution is layered over a region of high percent acrylamide during the electrophoresis media casting procedure. The lower region of the media that contains the high percentage acrylamide is referred to as the separating gel. The upper region of the media contains the lower percentage acrylamide and is generally referred to as the stacking gel. A distinct interface is generally formed between the separating and stacking gel regions. It is at this interface that a principal called "stacking" occurs during electrophoresis. Stacking enhances the separation and resolution of closely related samples that could not otherwise be achieved without the use of the stacking gel.
Polyacrylamide gels above 15% are extremely difficult to prepare. The polymerization reaction is extremely exothermic wherein the amount of heat generated increases with the percent of acrylamide. The heat generated by the polymerization reaction creates convection currents within the media that results in regions of polymer non-uniformity throughout the media. Bubbles are also formed within the media which further disrupt the media's uniformity and the media's ability to evenly sieve the molecules being separated.
Gradient polyacrylamide gels have been developed to combat these problems. Gradient polyacrylamide gels are gels where the percentage of polyacrylamide increases gradually from 5% at the top of the gel to 27% at the bottom of the gel. The heat generated within the relatively narrow 20-27% polyacrylamide region in the gradient gel can be effectively dissipated using fans or cooling manifolds to cool the cassette during polymerization to produce a satisfactory gel. Proteins in the 10,000-25,000 Dalton range can often be resolved within the narrow 27% polyacrylamide region at the bottom of the gradient gel.
A major problem effecting the stability and resolution of all electrophoresis media is water absorbtion and dissorption. Water absorbtion causes the pores within a given media to increase in size. As noted above, an electrophoresis media's sieving properties are largely governed by the media's pore size. Hence, when the media absorbs water, the media's pore sizes increase thereby enabling molecules to migrate across the media more rapidly. As a result, water absorbtion reduces the resolution achieved by the media since the degree of band separation achieved is reduced when the molecules move more rapidly across the media.
In addition to reducing the resolution (band separation) achieved in each lane of the media, water absorbtion is generally not uniform over the various portions of the media. Rather, water absorbtion in the center region of the electrophoresis media generally occurs at a faster rate than along the edges of the media. As a result, molecules located in the center lanes of the media move across the media at an accelerated rate as compared to the outer lanes. This undermines the reproducibility of the media. The effect water absorbtion has on the media's performance is illustrated in FIG. 1.
Band position comparisons are critical for assigning properties (e.g. molecular weight, net charge, structural characteristics) to a given sample. An effective means for controlling the rate at which water is absorbed by a medium is therefore essential to enhancing the stability and storageability of the media against the effects of water absorbtion.
Water absorbtion is a particularly significant problem in the case of high percentage polyacrylamide gels, "high percentage" referring to gels possessing greater than 20% acrylamide. All polyacrylamide gels absorb water during the polymerization process and during their subsequent storage. The tendency of polyacrylamide gels to absorb water increases with the percentage of acrylamide.
In stack buffer systems, the difference in acrylamide concentration between the stacking gel and the separating gel creates a significant osmotic potential between the two gels that causes water to move from the stacking gel (region of low percent acrylamide) to the separating gel (region of high percent acrylamide). The process of water diffusion from the stacking gel to the separating gel is very rapid and continues until an equilibrium is established between the stacking gel and the separating gel. As a result of water diffusion into the separating gel, current high percentage acrylamide stacking buffer systems are unstable and must be used within a day of their preparation. As a result of their instability, storable high percentage acrylamide gels are not presently commercially available.
When a high percentage acrylamide gel is stored in a buffer, the gel also rapidly absorbs water from the buffer until an equilibrium is established between the buffer and the gel. Unacceptable gels that exhibit a non-uniform pore size result from water diffusion between the high percentage acrylamide gel and air.
There is presently a need to be able to control the diffusion of water within electrophoresis media, between two electrophoresis media having different osmotic potentials, as is the case in multiple component electrophoresis media systems, between electrophoresis media and an accompanying buffer as well as between electrophoresis media and air. Once a method is developed to control the diffusion of water into and between electrophoresis media, the stability and storagability of electrophoresis media will be improved. Further, the reproducibility of the resulting media will be significantly enhanced.